Present imaging techniques for living cells and tissues are unable to produce accurate cell counts in cell clusters such as tissue samples and developing embryos past about eight cells in an aggregate, or past the eight cell stage of a living embryo non-invasively. Accurate cell counts of living embryos is important in the process of in vitro fertilization (IVF). In order to increase the success rate of IVF, doctors transfer multiple embryos back into the mother, with the transfer of three embryos having long been the standard. However, studies have shown that the transfer of two embryos does not decrease the overall ongoing pregnancy rate so there is a slow change toward reducing the standard. Even with the transfer of two embryos many IVF clinics report an overall pregnancy rate of only 30-40% (CDC, 2004; Gerris et al., 2002), with 20-40% of these successful births resulting in multiple pregnancies (Gerris et al., 2002; De Neubourg et al., 2002; ESHRE, 2001; Martin & Park, 1999). Multiple pregnancies increase the risks of cerebral palsy, preterm delivery, low birth weight, congenital malformations, and infant death (Gardner et al., 1995; Guyer et al., 1997; Kiely et al., 1998; Kinzler et al., 2000; Pharoah et al., 1996; Senat et al., 1998; Spellacy et al., 1990; Stromberg et al., 2002). Therefore, there is a worldwide effort to identify one healthy embryo for transfer back to the mother (Gurgan & Demirol, 2004).
One reason for the low success rate of IVF is the uncertainty of knowing whether an embryo is healthy enough to produce a successful pregnancy. To assess the quality of the embryo during the preimplantation period, the number of cells and the overall morphology of the embryo are recorded over the observation period (De Neubourg et al., 2002). Developments in embryo culture systems have allowed for viable embryos to develop in vitro into the blastocyst stage, consisting of more than 30 cells in a mouse embryo, on day 5 or 6 (Gardner & Lane, 1997). With the extended observation time, better decisions can be made on the quality of the embryo, and slightly better percentages of pregnancies have been recorded.
The rate of cell development during the one to eight cell stage is an important criterion for choosing a high quality embryo. However, available imaging technology makes obtaining this information non-invasively impossible past the eight cell stage. Fertility centers presently use differential interference contrast (DIC) microscopy to image embryos non-invasively. This method shows distinct cell boundaries within the focal plane when multiple cells do not lie in the path to the microscope objective. Some cell edges can be detected when only a couple cells overlap because embryos are optically transparent, allowing two layers of four cells to be imaged. However, cell boundaries cannot be detected for multiple layers of cells, making it impossible to produce accurate cell counts past the eight cell stage with DIC. As a result of insufficient imaging capabilities, the criteria for a high quality embryo at the blastocyst stage are primarily based on the morphology of the sample and the rate of development. Therefore, there is a need for an instrument that can non-invasively count the number of cells during embryo development up to the blastocyst stage in order to help physicians make better determinations regarding embryo quality. The availability of such an instrument would increase the success of single embryo transfer and reduce the motivation for multiple embryo transfers.